Epithelial-stromal interactions are thought to play a critical role in maintaining normal corneal homeostasis, and in regulation of corneal wound healing following injury and refractive surgery. For example, following PRK, injured epithelial cells release several cytokines and growth factors that modulate stromal cell death, proliferation, and differentiation. While the importance of the cross-talk between corneal epithelial and stroma cells is clear, a more detailed investigation of the role of specific cytokines in mediating epithelial-stromal interactions requires an in vitro model in which culture conditions can be tightly regulated. Unfortunately, current in vitro models do not adequately mimic the in vivo 3-D geometry and mechanical properties of corneal tissue. Two of the key barriers to successful tissue engineering of an in vitro anterior corneal construct are: (1) the development of a 3-D extracellular matrix which has high mechanical stiffness, yet supports maintenance of the quiescent stromal cell (keratocyte) phenotype, and (2) the development of a corneal epithelial cell line that differentiates and stratifies in vitro. We have recently performed pilot experiments in which cell-seeded 3-D collagen matrices were compressed to achieve high stiffness tissue equivalents. These constructs support differentiation of corneal keratocytes, and have several unique properties which may be ideal for stromal tissue engineering. We have also developed a unique human telomerase-immortalized corneal epithelial cell line (hTCEpi), which expresses key differentiation markers under stratified, air-lifted culture conditions. The purpose of the proposed research is to test whether we can combine these two unique technologies to produce a novel 3-D model that can be used for studying epithelial-stromal interactions during homeostasis and wound healing. To accomplish this, we propose to: (1) determine whether compressed collagen matrices can be used to support the differentiation and stratification of corneal epithelium under serum-free conditions in vitro, and (2) investigate the feasibility of inducing injuries to these constructs in order to simulate anterior corneal wound healing. Accomplishing these aims would establish the overall feasibility of using compressed collagen matrices as a model system for studying epithelial-stromal interactions during homeostasis and wound healing, and thus provide a foundation for more detailed studies in which the specific factors mediating these critical interactions can be investigated. PUBLIC HEALTH RELEVANCE: Epithelial-stromal interactions play a critical role in normal corneal function and in regulation of corneal wound healing following injury and refractive surgery. The goal of the proposed research is to develop a novel, multi-layered 3-dimensional culture model that can be used to investigate these interactions under controlled, in vitro conditions.